Phenols

DOC316.53.01108

USEPA1 4-Aminoantipyrine Method2

Method 8047

0.002 to 0.200 mg/L

Scope and application: For water and wastewater.
1
2

USEPA accepted (distillation required); procedure is equivalent to USEPA method 420.1 for wastewater.
Adapted from Standard Methods for the Examination of Water and Wastewater.

Test preparation
Instrument-specific information
Table 1 shows all of the instruments that have the program for this test. The table also
shows sample cell and orientation requirements for reagent addition tests, such as
powder pillow or bulk reagent tests.
To use the table, select an instrument, then read across to find the applicable information
for this test.
Table 1 Instrument-specific information
Instrument

Sample cell orientation

Sample cell

DR 6000

The fill line is to the right.

2612602

DR 3800
DR 2800
DR 2700
DR 1900
DR 5000

The fill line is toward the user.

DR 3900

Before starting
Analyze samples within 4 hours to prevent oxidation.
Spilled reagent can have an effect on the test results and is hazardous to skin and other materials.
Use chloroform only with proper ventilation.
The Phenol 2 Reagent Powder Pillows contain potassium nitroferricyanide. Keep cyanide solutions at pH > 11 to prevent
exposure to hydrogen cyanide gas. Collect the reacted samples for proper disposal.
Do not pour chloroform solutions down the drain. Collect the water that is saturated with chloroform, chloroform
solutions and the cotton plug used in the delivery tube of the separatory funnel for proper disposal. Refer to a current
MSDS/SDS for safe handling and disposal instructions.
Make the cotton plug the size of a pea. A larger plug restricts the flow and a smaller plug can dislodge from the delivery tube
of the funnel.
In bright light conditions (e.g., direct sunlight), close the cell compartment, if applicable, with the protective cover during
measurements.
Review the Safety Data Sheets (MSDS/SDS) for the chemicals that are used. Use the recommended personal protective
equipment.
Dispose of reacted solutions according to local, state and federal regulations. Refer to the Safety Data Sheets for disposal
information for unused reagents. Refer to the environmental, health and safety staff for your facility and/or local regulatory
agencies for further disposal information.

Items to collect
Description

Quantity

Chloroform, ACS

60 mL

Phenol Reagent Powder Pillow

Phenol 2 Reagent Powder Pillow

Clippers

Cotton balls

varies

Cylinder, graduated, 50-mL

Cylinder, graduated, 500-mL

Funnel, 500 mL separatory with stand and stopper

Hardness 1 Buffer Solution, pH 10.1

10 mL

Pipet, volumetric, Class A, 5.00-mL

Ring, support, 4-in.

Support for ring stand, 5 x 8 in. base

Sample cells (For information about sample cells, adapters or light shields, refer to Instrumentspecific information on page 1.)

Water, deionized

varies

Refer to Consumables and replacement items on page 6 for order information.

Sample collection and storage

For the most reliable results, analyze the samples within 4 hours after collection. Use the
storage instructions that follow if prompt analysis is not possible:
1.
2.
3.
4.
5.

Collect 500 mL of the sample in a clean glass container.

Add the contents of two Copper Sulfate Powder Pillows.
Adjust the pH to 4 or less with 10% Phosphoric Acid Solution.
Keep the preserved samples at or below 6 C (43 F) for a maximum of 24 hours.
Analyze the preserved samples within 24 hours.

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

Test procedure

Start

1. Start program 470

Phenol. For information
about sample cells,
adapters or light shields,
refer to Instrument-specific
information on page 1.
Note: Although the program
name can be different
between instruments, the
program number does not
change.

2. Measure 300 mL of
deionized water in a 500-mL
graduated cylinder.

3. Prepare the blank: Pour

the measured deionized
water into a 500-mL
separatory funnel.

4. Measure 300 mL of the

sample in a 500-mL
graduated cylinder.

5. Prepare the sample:

Pour the measured sample
into another 500-mL
separatory funnel.

6. Add 5 mL of Hardness
Buffer to each separatory
funnel.

7. Put the stoppers on the

funnels. Shake to mix.

8. Add the contents of one

Phenol Reagent Powder
Pillow to each separatory
funnel.

9. Put the stoppers on the

funnels. Shake to dissolve
the reagent.

10. Add the contents of one

Phenol 2 Reagent Powder
Pillow to each separatory
funnel.

11. Put the stoppers on the

funnels. Shake to dissolve
the reagent.

12. Add 30 mL of
chloroform to each
separatory funnel.

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

13. Put the stoppers on the

funnels. Invert each funnel
and open the stopcock to
vent. Close the stopcocks,
shake each funnel briefly,
then invert and open the
stopcock to vent.

14. Vigorously shake each

funnel for 30 seconds. Open
the stopcock to vent if
necessary.

15. Remove the stoppers.

Wait for the layers to
separate. If there is phenol
in the sample, the bottom
chloroform layer will have a
yellow to amber color.
Note: Do the rest of the test
procedure quickly because
the chloroform will
evaporate, which causes
high readings.

16. Put a cotton plug the

size of a pea into the
delivery tube of each funnel
to remove suspended water
or particles from the
chloroform.

Zero

17. Drain the chloroform

layers into separate sample
cells (one for the blank and
one for the sample). Put the
stopper on the cells. The
volume of chloroform extract
is approximately 25 mL.
The water phase in the
funnel contains chloroform.
Make sure to dispose of the
solution properly.

18. Clean the blank sample

cell.

19. Insert the blank into the

cell holder.

20. Push ZERO. The

display shows 0.000 mg/L
Phenol.

Read

21. Clean the prepared

sample cell.

22. Insert the prepared

sample into the cell holder.

23. Push READ. Results

show in mg/L Phenol.

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

Interferences
Interfering substance

Interference level

pH

The sample pH must be between 3 and 11.5 for best results.

Oxidizing or reducing agents

Can interfere. Distill the samples (Refer to Distill the sample

on page 5).

Sulfides or suspended matter

Distillation or the following pretreatment is necessary:

1.

2.
3.

Measure 350 mL of sample with a clean 500-mL

graduated cylinder. Pour the sample into a clean 500-mL
Erlenmeyer flask.
Add the contents of one Sulfide Inhibitor Reagent
Powder Pillow. Swirl to mix.
Filter 300 mL of the sample through a folded filter paper.
Use this solution in step 4 of the procedure.

Distill the sample

To remove interferences, use the steps that follow to distill the sample. For the best
results, make sure that the sample pH is between 3 and 11.5. Refer to Interferences
on page 5 for pretreatment guidelines.
1. Set up the distillation apparatus for general purpose distillation. Refer to the
Distillation Apparatus manual for proper assembly.
2. Set up a 500-mL Erlenmeyer flask to collect the distillate. It may be necessary to use
a laboratory jack to elevate the flask.
3. Measure 300 mL of water sample in a clean 500-mL graduated cylinder. Pour the
sample into the distillation flask.
Note: For proof of accuracy, use a 0.200-mg/L phenol standard in addition to the sample.
Refer to Accuracy check on page 5.

4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

Add a magnetic stir bar and 5 glass beads.

Use a serological pipet to add 1 mL of Methyl Orange Indicator to the distillation flask.
Set the stirrer power to on. Set the stir control to 5.
Add 10% Phosphoric Acid Solution drop-wise until the indicator changes from yellow
to orange.
Add one Copper Sulfate Powder Pillow to the distillation flask. Let the reagent
dissolve. Do not do this step if copper sulfate is used to preserve the sample.
Put the cap on the distillation flask.
Turn on the water and adjust to maintain a steady flow through the condenser.
With the thermometer inserted, set the heat control to 10. The yellow pilot lamp is an
indication that the heater is on.
Collect 275 mL of distillate in the Erlenmeyer flask, then set the heat to off.
Fill a 25-mL graduated cylinder to the 25-mL mark with deionized water.
Add the water to the distillation flask.
Set the heat control to 10. Heat until another 25-mL of distillate is collected.
Use a clean graduated cylinder to measure the distillate again to make sure that
300 mL was collected.
Use the distillate in the test procedure.

Accuracy check
Standard solution method
Items to collect:

Phenol, ACS

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

1000-mL volumetric flasks (2), Class A

500-mL volumetric flask, Class A
10-mL volumetric pipet, Class A, with safety bulb
Deionized water

1. Prepare a 1000-mg/L phenol stock solution as follows:

a. Weigh 1.000 g of phenol.
b. Add the phenol to a 1000-mL volumetric flask.
c. Dilute to the mark with freshly boiled and cooled deionized water. Mix well to
dissolve.
2. Prepare a 10-mg/L working phenol standard solution as follows:
a. Use a pipet to add 10 mL of the stock phenol solution to a 1000-mL volumetric
flask.
b. Dilute to the mark with deionized water. Mix well.
3. Prepare a 0.200-mg/L standard solution as follows:
a. Use a pipet to add 10 mL of the 10-mg/L working solution to a 500-mL volumetric
flask.
b. Dilute to the mark with deionized water. Mix well.
4. Use the test procedure to measure the concentration of the prepared standard
solution.
5. Compare the expected result to the actual result.
Note: The factory calibration can be adjusted slightly with the standard adjust option so that the
instrument shows the expected value of the standard solution. The adjusted calibration is then
used for all test results. This adjustment can increase the test accuracy when there are slight
variations in the reagents or instruments.

Method performance
The method performance data that follows was derived from laboratory tests that were
measured on a spectrophotometer during ideal test conditions. Users can get different
results under different test conditions.
Program

Standard

Precision (95% confidence interval)

Sensitivity
Concentration change per 0.010 Abs change

470

0.100 mg/L Phenol

0.0930.107 mg/L Phenol

0.002 mg/L Phenol

Summary of Method
The 4-aminoantipyrine method measures all ortho- and meta-substituted phenols. These
phenols react with 4-aminoantipyrine in the presence of potassium ferricyanide to form an
antipyrine dye. The dye is removed from the aqueous phase by extraction with
chloroform. The sensitivity of the method changes with the type of phenolic compound.
Because water samples can contain various types of phenolic compounds, the procedure
results are shown as the equivalent concentration of phenol. The measurement
wavelength is 460 nm.

Consumables and replacement items

Required reagents
Description

Quantity/Test

Unit

Item no.

2243900

Chloroform, ACS

60 mL

4L

1445817

Hardness 1 Buffer Solution, pH 10.1

10 mL

500 mL

42449

100/pkg

87299

Phenols Reagent Set (100 Tests), includes:

Phenol Reagent Powder Pillows

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

Consumables and replacement items (continued)

Description

Quantity/Test

Unit

Item no.

100/pkg

183699

varies

4L

27256

Quantity/test

Unit

Item no.

Clippers

each

96800

Cotton balls, absorbent

100/pkg

257201

Cylinder, graduated, 50-mL

each

50841

Cylinder, graduated, 500-mL

each

50849

Funnel, separatory, 500-mL

each

52049

Pipet filler, safety bulb

each

1465100

Pipet, volumetric, Class A, 5.00-mL

each

1451537

Support Ring, 4-inch

each

58001

Support, Ring Stand, 5-inch x 8-inch base

each

56300

Description

Unit

Item no.

Balance, analytical, 80 g x 0.1 mg 100240 VAC

each

2936701

50/pkg

1481866

Distillation heater and support for apparatus set, 115 VAC option

each

2274400

Distillation heater and support for apparatus set, 230 VAC option

each

2274402

Distillation apparatus set, general purpose

each

2265300

Filter paper, folded, 12.5-cm

100/pkg

189457

Flask, Erlenmeyer, 500-mL

each

50549

Funnel, poly, 65-mm

each

108367

100 mL MDB

14832

113 g

75814

100 mL MDB

1476932

Sulfide Inhibitor Reagent Powder Pillows

100/pkg

241899

Paper, pH, 014 pH range

100/pkg

2601300

each

2635700

Phenol 2 Reagent Powder Pillows

Water, deionized

Required apparatus
Description

Distillation reagents and apparatus

Copper Sulfate Powder Pillows

Methyl Orange Indicator Solution, 0.5-g/L

Phenol, ACS
Phosphoric Acid Solution, 10%

Thermometer, non-mercury, 10 to +225 C

Pipet, TenSette , 1.010.0 mL

each

1970010

250/pkg

2199725

50/pkg

2199796

Safety goggles, vented

each

2550700

Gloves, chemical resistant, size 99.5

pair

24101041

Flask, volumetric, Class A, 500-mL glass

each

1457449

Flask, volumetric, Class A, 1000-mL glass

each

1457453

Pipet tips for TenSette Pipet, 1.010.0 mL

Pipet tips for TenSette Pipet, 1.010.0 mL

Other sizes available

Phenols, 4-Aminoantipyrine Method (0.200 mg/L)

FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING:

In the U.S.A. Call toll-free 800-227-4224
Outside the U.S.A. Contact the HACH office or distributor serving you.
On the Worldwide Web www.hach.com; E-mail techhelp@hach.com

Hach Company/Hach Lange GmbH, 2007, 2010, 2012, 2014. All rights reserved.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932

03/2014, Edition 8